Comparison of intracellular signalling by insulin and the hypermitogenic AspB10 analogue in MCF-7 breast adenocarcinoma cells

We compared mitogenicity and intracellular signalling by human insulin and the AspB10 (X‐10) human insulin analogue in MCF‐7 human mammary adenocarcinoma cells. By flow analysis of phosphorylated histone H3 or cell cycle distributions, insulin and X‐10 were mitogenic at physiologically relevant concentrations (2 nm to 74 pm range), with X‐10 being approximately 3‐fold more mitogenic than insulin. By western blotting with phospho‐specific antibodies, insulin induced phosphorylation of IRS‐1, Akt, p70S6K, S6 ribosomal protein, 4E‐BP1, FoxO3a, FoxO1, p44/42 MAPK and the EGFR. Blocking with wortmannin, rapamycin and U0126 showed that these signalling events conformed to the canonical PI3K pathway. IRS‐1 (Ser302) phosphorylation was abolished by wortmannin and rapamycin, suggesting a feedback from the PI3K pathway on insulin signalling. Compared with equimolar insulin, X‐10 caused up to 2‐fold higher phosphorylation of all proteins examined in this study. The phosphorylation sites that responded most strongly to insulin were not generally the same as those responding most strongly to X‐10. In the PI3K pathway, the most X‐10‐sensitive protein localized to the translation‐regulating arm (p70S6K), with FoxO3a and FoxO1 transcription factors showing a more comparable response to insulin and X‐10. Using flow analysis, we confirmed the correlation between insulin‐triggered translational activation in G0/G1 (S6 phosphorylation) and S‐phase entry by MCF‐7 cells. In summary, our findings implicate asymmetrical PI3K pathway activation and specifically stimulation of protein translation in the hypermitogenic effect of insulin analogues such as X‐10. It remains to be shown whether these findings are relevant to other human mammary cancer cell types. Copyright © 2010 John Wiley & Sons, Ltd. In an optimized MCF‐7 human mammary adenocarcinoma system, insulin and X‐10 were mitogenic already at concentrations from 74 pM, with X‐10 being approximately 3‐fold more mitogenic than insulin. Compared to equimolar insulin, X‐10 caused up to 2‐fold higher phosphorylation of all 11 proteins examined in this study, the most X‐10‐sensitive protein being p70S6K. In summary, our findings implicate asymmetrical PI3K pathway activation and specifically stimulation of protein translation in the hypermitogenic effect of insulin analogues such as X‐10.